Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp.

Abstract

Stereoselective reduction towards pharmaceutically potent products with multi-chiral centers is an ongoing hot topic, but up to now catalysts for reductions of bulky aromatic substrates are rare. The NADPH-dependent alcohol dehydrogenase from Ralstonia sp. (RADH) is an exception as it prefers sterically demanding substrates. Recent studies with this enzyme indicated outstanding potential for the reduction of various alpha-hydroxy ketones, but were performed with crude cell extract, which hampered its detailed characterization. We have established a procedure for the purification and storage of RADH and found a significantly stabilizing effect by addition of CaCl(2). Detailed analysis of the pH-dependent activity and stability yielded a broad pH-optimum (pH 6-9.5) for the reduction reaction and a sharp optimum of pH 10-11.5 for the oxidation reaction. The enzyme exhibits highest stability at pH 5.5-8 and 8-15°C; nevertheless, biotransformations can also be carried out at 25°C (half-life 80 h). Under optimized reaction parameters a thorough study of the substrate range of RADH including the reduction of different aldehydes and ketones and the oxidation of a broad range of alcohols was conducted. In contrast to most other known alcohol dehydrogenases, RADH clearly prefers aromatic and cyclic aliphatic compounds, which makes this enzyme unique for conversion of space demanding substrates. Further, reductions are catalyzed with extremely high stereoselectivity (>99% enantio- and diastereomeric excess). In order to identify appropriate substrate and cofactor concentrations for biotransformations, kinetic parameters were determined for NADP(H) and selected substrates. Among these, we studied the reduction of both enantiomers of 2-hydroxypropiophenone in more detail.

Structures of Alcohol Dehydrogenases from Ralstonia and Sphingobium spp. Reveal the Molecular Basis for Their Recognition of ‘Bulky–Bulky’ Ketones

“…During this process problems with precipitation were encountered, but these were successfully addressed through the inclusion of Ca 2+ ions, 500 mM sodium chloride and glycerol in the cell resuspension and protein purification buffers (see Experimental Section). Gel filtration studies on the protein derived in this way were suggestive of a tetramer of RasADH monomers in solution, rather than the trimer suggested by earlier studies [9].…”

“…The model suggest that while the flexible alkyl chain of 1 can be accommodated in the distal region of hydrophobic tunnel through contortion in RasADH, less flexible substituents, such as aromatics, would not be accommodated. This is supported by substrate specificity studies [9], which show that compounds such as benzoin are poor substrates for RasADH. For SyADH, however, the larger distal portion of the hydrophobic tunnel may allow for the accommodation of such bulky groups.…”

“…The excellent enantioselectivity was also extended to bulky-small ketones such as acetophenone. RasADH has subsequently been characterised in some detail by one of our groups [9]. The enzyme was reported to be of the short chain dehydrogenase (SDR) family, with a subunit molecular weight of 26.7 kDa (249 amino acids) and it was suggested that three subunits of the enzyme associated to form a trimer in solution.…”

Purification and characterization of a novel glutamate dehydrogenase from Geotrichum candidum with higher alcohol and amino acid activity

“…The native-PAGE and HPLC analyses indicated that the enzyme was composed of 4 monomeric subunits. This is different from all currently reported higher alcohol-reducing enzymes that typically have subunits that have dimolecular weights of 26–80 kDa (Kataoka et al 2006; Kulig et al 2013; Yamada-Onodera et al 2007). By examination of the phylogenetic tree of the NAD(P)-dependent ADH family of proteins and related amino acid sequences in the existing protein database (NCBI) (Jeon et al 2008), the G. candidum S12 GDH obtained in this study may exhibit alcohol dehydrogenase and GDH (EC1.4.1.3) activity for activity towards glutamate and higher alcohols, respectively.…”

Enantioselective Reduction of Ethyl 3‐Oxo‐5‐phenylpentanoate with Whole‐Cell Biocatalysts

“…Conversely, the ADH from Ralstonia sp. [22,23] ( Table 1, Entry 1) afforded the desired (S) product with moderate yield (54 % on [a] Conditions: 10 mg of lyophilized biocatalyst, glucose dehydrogenase (1 mg mL -1 ), 20 mM glucose, 0.5 mM NADH, 0.5 mM NADPH, 10 mM substrate in 500 μL reaction mixture Tris-HCl buffer pH 7.5/2-propanol (90:10, v/v), 30°C, 120 rpm in a thermoshaker for 20 h. Conversion (i.e., consumption of substrate) and product yield (i.e., formation of 1) were determined by HPLC analysis on an achiral stationary phase by using calibration curves for 1 and 2. [a] Conditions: 10 mg of freeze-dried E. coli cells containing the recombinant ADH, glucose dehydrogenase (1 mg mL -1 ), 20 mM glucose, 0.5 mM NADH, 0.5 mM NADPH, 10 mM substrate in 500 μL reaction mixture Tris-HCl buffer pH 7.5/2-propanol (90:10, v/v), 30°C, 120 rpm in a thermoshaker for 20 h. Conversion (i.e., consumption of substrate) and product yield (i.e., formation of 1) were determined by HPLC analysis on an achiral stationary phase by using calibration curves for 1 and 2.…”